Bariloche protein symposium argentine society for biochemistry and molecular biology

31
BIOCELL, 27 (Suppl. I), 2003
L7.
MOLECULAR REGULATION OF MEMBRANE TRAFFIC
BETWEEN THE ER AND THE GOLGI
Elizabeth Sztul.
Department of Cell Biology.  University of Alabama at
Birmingham.  Birmingham, AL.  USA. E-mail: [e-mail]
Protein transfer between membrane bound compartments of
eukaryotic cells occurs by membrane traffic. Proteins traverse the
secretory pathway through progressive steps of vesicle formation,
movement, targeting and fusion. Over the last decade, significant
insight has been gained into traffic from the Endoplasmic
Reticulum (ER) to the Golgi, the first “membrane station” in the
secretory pathway.  It is now apparent that proteins exit the ER at
specialized subdomains called ER exit sites. Vesicles bud from
ER exit sites and form new compartments called the vesiculo-
tubular clusters (VTCs). VTCs become transport-competent after
they are remodeled by the removal of select proteins. VTCs then
attach to microtubules and in a motor-mediated process move to
the Golgi region where they fuse. One of the key challenges in
cell biology is to uncover the spatial and temporal sequence of
events that occur at each step. The progress has been on two fronts:
the description of the compartments through which proteins move,
and the identification and functional characterization of molecules
that mediate traffic. Among them are small GTPases, activators
and inactivators of those GTPases, COPII and COPI coat
components, tethering factors and SNAREs. Despite significant
progress, active investigation continues to uncover the relationships
between these molecules. This lecture will highlight some of the
molecular events regulating ER to Golgi transport.
L8.
MOLECULAR MECHANISMS REGULATING CELL
CYCLE EXIT
Sergio Moreno Perez (Spain)
L9.
PEPTIDE SEQUENCING BY PSD MALDI-TOF MS USING
CAF METHODOLOGY
Ulf Hellman.
Ludwig Institute for Cancer Research, Uppsala Branch, Sweden.
L10.
RNA AND DNA VIRUS CAPSIDS AS NANO PLATFORMS
AND NANO MACHINES
John E. Johnson.
Department of Molecular Biology, MB31, The Scripps Research
Institute, La Jolla, CA, 92037. E-mail: [e-mail]
Viruses are two-edged swords. Many are dangerous pathogens that
cause extraordinary human suffering, mortality and economic loss
(e.g HIV and SARS), but benign viruses are now recognized as
viable reagents for applications in nano technology, chemistry and
biology. We used a static, icosahedral, 30nM plant virus as an
addressable "nano block" for molecular electronics, protein
immobilization, and novel particle patterning on gold surfaces.
RNA and DNA virus capsids that exhibit large-scale, pH sensitive,
protein reorganization have recently been mechanistically
characterized by biophysics and molecular genetics for the
harnessing of these properties for nano devices. The characteristics
of these remarkable nano machines will be presented and potential
applications discussed.
- Wang Q, Lin T, Johnson J, Finn M (2002). Icosahedral Virus Particles
as Addressable Nanoscale Building Blocks. Angew Chem Int Ed. 41: 459-
462.
- Canady M, Tsuruta H, Johnson J (2001). Analysis of Rapid, Large-Scale
Protein Quaternary Structural Changes: Time-Resolved X-ray Solution
Scattering of Nudaurelia capensis w Virus (NwV) Maturation. J Mol Biol
311: 803-814.
- Conway J, Wikoff W, Cheng N, Duda R, Hendrix R, Johnson J, Steven A
(2001). Virus maturation via large subunit rotations and local refolding.
Science 292: 744-748.

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32
BIOCELL, 27 (Suppl. I), 2003
S1.
STRUCTURAL BASIS OF GLYCOGEN SYNTHESIS
Pedro M. Alzari, Alejandro Buschiazzo, Marcelo E. Guerin, Juan
E. Ugalde
1
 and Rodolfo A. Ugalde
1
.
Institut Pasteur, Paris, France; and 
1
IIB, UNSAM, Argentina.
E-mail: [e-mail]
Glycogen and starch are the major carbon and energy storage
compounds in most living organisms. The understanding of
glycogen metabolism is an important subject in general
biochemistry.  Glycogen synthase [EC 2.4.1.21] catalyzes the
addition of individual glucosyl subunits to the growing chain of
glycogen. It is a key component of the enzymatic machinery
involved in glycogen metabolism, together with glycogen
phosphorylase and the branching/debranching enzymes. Glycogen
synthases from bacteria and higher plants (starch synthases) are
α-retaining family 5 glycosyl transferases (for a classification of
glycosyl transferases, see http://afmb.cnrs-mrs.fr/CAZY) that use
ADP-glucose as sugar donor and have MW around 50 KDa.
Mammalian and yeast GSs belong to family 3 glycosyl transferases,
are larger enzymes (~80 KDa) and prefer UDP-glucose to ADP-
glucose as the sugar donor. We now present the 3D structure of a
bacterial GS at 2.3 Å resolution. The recombinant enzyme from
Agrobacterium tumefaciens was purified to homogeneity and
crystallized. The structure was determined by single-wavelength
anomalous diffraction methods, revealing a two-domain 
α/β
folding topology. Crystals of GS in complex with specific ligands
identified the catalytic center and its architecture strongly suggest
that glycogen synthase and glycogen phosphorylase are
evolutionary related enzymes, indicating that glycogen is
synthesized and degraded by homologous enzymes. The
implications of the 3D structure in terms of protein folding,
catalytic mechanism and activity regulation will be discussed.
S2.
THE MULTI-FACETED MANNOSE 6-PHOSPHATE
RECEPTORS
N.M. Dahms.
Medical College of Wisconsin.USA. E-mail: [e-mail]
The 46kDa cation-dependent mannose 6-phosphate receptor (CD-
MPR) and the 300kDa cation-independent mannose 6-phosphate/
insulin-like growth factor II (IGF-II) receptor (CI-MPR) are the
sole members of the P-type lectin family and are distinguished
from all other lectins by their ability to recognize phosphorylated
mannose residues. These receptors play an essential role in the
generation of functional lysosomes within the cells of higher
eukaryotes by directing newly synthesized lysosomal enzymes
bearing the mannose 6-phosphate (M6P) signal from the trans
Golgi network to lysosomes. The CI-MPR has been implicated in
several other processes, including cell growth, apoptosis, and cell
migration, due to its ability to bind a wide range of M6P-containing
(e.g., latent transforming growth factor-beta, granzyme B,
proliferin) and non-M6P-containing (IGF-II, retinoic acid,
urokinase-type plasminogen activator receptor (uPAR),
plasminogen) molecules at the cell surface.  The ability of the CI-
MPR to interact with many different proteins and a lipophilic
molecule is facilitated by the receptor ’s ~2,270-residue
extracytoplasmic region comprised of 15 homologous domains in
which several binding sites have been localized to individual
domains. Our studies have provided a detailed view of the
mechanism of high affinity (nM) phosphomonoester recognition
by both MPRs. Our recent structural studies have provided insights
into the nature of plasminogen and uPAR recognition by the CI-
MPR and has allowed us to generate a model of the entire
extracytoplasmic region that provides a context with which to
envision the numerous binding interactions carried out by this
multi-faceted receptor.
S3.
THE NUCLEOTIDE SUGAR TRANSPORT/ANTIPORT
CYCLE OF THE ENDOPLASMIC RETICULUM AND
GOLGI APPARATUS: FROM BASIC SCIENCE TO
DISEASE
Carlos B. Hirschberg.
Boston University. USA. E-mail: [e-mail]
Approximately half of the proteins in eukaryotes are either
membrane bound or secreted. Eighty percent undergo
posttranslational modifications, such a glycosylation, sulfation and
phosphorylation in the lumen of the endoplasmic reticulum and
Golgi. Nucleotide sugars, nucleotide sulfate and ATP are substrates
for these reactions and must first be transported from the cytosol
into the lumen of the above organelles. This transport is coupled
to the exchange with the corresponding nucleoside monophosphate,
an antiport. We have purified, reconstituted into liposomes and
cloned several transporters of the Golgi as well as the enzymes
responsible for generating the antiporter nucleoside
monophosphate molecules. While transporters are
multitransmembrane spanning proteins nucleotide disphosphatases
are type II membrane proteins. Proteins of very different amino
acid sequences may transport the same substrates. Genetic studies
have shown that mutations or deletion of transporter or
diphosphatase genes result in defects of glycosylation of proteins
and lipids. In C. elegans we found that several transporters of
dual substrate specificities have very different protein sequences,
cell type differential expression and biological functions. One of
the nucleotide diphosphatase mRNAs is upregulated by conditions
leading to endoplasmic reticulum stress suggesting that it plays a
role in quality control of glycoprotein folding. . Finally a disease
has been recently described in which the Golgi apparatus GDP-
fucose transporters is partially defective leading to severe  growth
and developmental impairments.
S4.
DOMAIN ORGANIZATION AND PATTERN
RECOGNITION OF UDP-GLC:GLYCOPROTEIN
GLUCOSYL TRANSFERASE (GT)
Julio J. Caramelo, Olga A. Castro, Gonzalo de Prat-Gay and
Armando J. Parodi.
Fundación Instituto Leloir, Buenos Aires, Argentina. E-mail:
[e-mail]
GT is the key element of the quality control of glycoprotein folding
occurring in the endoplasmic reticulum (ER). The enzyme only
glucosylates glycoproteins not displaying their native structures.
Structures created by GT (monoglucosylated N-glycans) are
specifically recognised by two ER resident lectins (calnexin and
calreticulin) thus precluding transit of not properly folded
glycoproteins to the Golgi. GT proved to be formed by at least two
tightly associated domains, the C-terminal or catalytic domain
(20% of the molecule) and the N-terminal, presumably involved
in conformation recognition. In spite of showing a poor similarity
at the primary sequence level (16.3%), the GT N-terminal domains
derived from two different species could be functionally
interchanged. Using chemically glycosylated derivatives of
chymotrypsin inhibitor 2 displaying several truncations from the
N-terminus, and thus different conformations, as GT substrates
we determined that GT recognises hydrophobic amino acid patches
exposed in molten globule-like conformations. Moreover, the
enzyme was able to differentially glucosylate conformers showing
minor structural differences.

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